title: Sequential assembly of an active RNA polymerase molecule at the air-water interface.
abstract: At the heart of understanding cellular processes lies our ability to explore the specific nature of communication between sequential information carrying biopolymers. However, the data extracted from conventional solution phase studies may not reflect the dynamics of communication between recognized partners as they occur in the crowded cellular milieu. We use the principle of immobilization of histidine-tagged biopolymers at a Ni(II)-encoded Langmuir monolayer to study sequence-specific protein-protein interactions in an artificially crowded environment. The advantage of this technique lies in increasing the surface density of one of the interacting partners that allows us to study macromolecular interactions in a controlled crowded environment, but without compromising the speed of the reactions. We have taken advantage of this technique to follow the sequential assembly process of the multiprotein complex Escherichia coli RNA polymerase at the interface and also deciphered the role of one of the proteins, omega (ω), in the assembly pathway. Our reconstitution studies indicate that in the absence of molecular chaperones or other cofactors, omega (ω) plays a decisive role in refolding the largest protein beta prime (β') and its recruitment into the multimeric assembly to reconstitute an active RNA polymerase. It was also observed that the monolayer had the ability to distinguish between sequence-specific and -nonspecific interactions despite the immobilization of one of the biomacromolecules. The technique provides a universal two-dimensional template for studying protein-ligand interactions while mimicking molecular crowding.
authors.affiliation: Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, India.
authors.forename: Abantika
authors.initials: A
authors.lastname: Ganguly
authors.forename: Dipankar
authors.initials: D
authors.lastname: Chatterji
mesh_terms: D000388:Air; D015153:Blotting, Western; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry*
chemical_list: D014867:Water; D012321:DNA-Directed RNA Polymerases